Development of SARS-CoV-2 infection in patients with rheumatic conditions on hydroxychloroquine monotherapy vs. patients without rheumatic conditions: A retrospective, propensity-matched cohort study.

BACKGROUND: The primary purpose of the current study was to examine whether patients with rheumatologic conditions receiving only chronic hydroxychloroquine therapy for their disease are at less risk of developing SARS-CoV-2 infection than a comparative group of patients without rheumatologic conditions. METHODS: A retrospective, observational, nationwide stratified propensity analysis was conducted comparing patients only on chronic treatment with hydroxychloroquine for their rheumatologic condition to a random sample of patients without rheumatologic conditions and not receiving hydroxychloroquine, utilizing a Veterans Health Administration nationwide clinical administrative database. RESULTS: The 1-to-1 stratified propensity analysis was undertaken using a random sample of patients without rheumatoid conditions and not receiving hydroxychloroquine (n  33,081) and patients with rheumatoid conditions receiving hydroxychloroquine as the lone medication for their condition (n  6047). A total of 5,474 patients in each group were successfully matched. The incidence of documented SARS-CoV-2 infections during the study period did not differ between patients receiving hydroxychloroquine and patients not receiving hydroxychloroquine (41/5,474 [0.749%] vs. 36/5,474 [0.658%], respectively, p = 0.57; Odds ratio [OR] 1.14, 95% confidence interval [CI] 0.73-1.79). There were no statistically-significant differences in secondary outcomes between the two groups in patients who developed active SARS-CoV-2 infection. Multivariate logistic regression to determine independent variables associated with the development of active SARS-CoV-2 infection failed to include receipt of hydroxychloroquine (OR 0.99, 95% CI 0.62-1.56). CONCLUSIONS: Hydroxychloroquine failed to demonstrate a preventative effect against SARS-CoV-2 infection in a large group of patients with rheumatologic conditions compared to patients without rheumatologic conditions.

Long-term hydroxychloroquine use in patients with rheumatic conditions and development of SARS-CoV-2 infection: a retrospective cohort study.

BACKGROUND: Hydroxychloroquine is one of several agents being evaluated in the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We aimed to examine whether patients with rheumatological conditions receiving chronic hydroxychloroquine therapy are at less risk of developing SARS-CoV-2 infection than those not receiving hydroxychloroquine. METHODS: This retrospective cohort study included de-identified information of all veterans in the US Veterans Health Administration clinical administrative database aged 18 years or older with rheumatoid arthritis, systemic lupus erythematosus, or associated rheumatological conditions (based on International Classification of Diseases, 10th edition, diagnostic codes) who were alive on March 1, 2020. A propensity score was calculated for each patient, and each patient who was receiving hydroxychloroquine was matched to two patients who were not receiving hydroxychloroquine (controls). The primary endpoint was the proportion of patients with PCR-confirmed SARS-CoV-2 infection among those receiving chronic hydroxychloroquine versus the propensity-matched patients not receiving chronic hydroxychloroquine between March 1 and June 30, 2020. Secondary outcomes were hospital admission associated with SARS-CoV-2 infection; intensive care requirement associated with SARS-CoV-2 infection; mortality associated with SARS-CoV-2 infection; and overall rates of any hospital admission and mortality (ie, all cause). Multivariate logistic regression analysis was done to determine independent variables for the development of active SARS-CoV-2 infection. FINDINGS: Between March 1 and June 30, 2020, 10 703 patients receiving hydroxychloroquine and 21 406 patients not receiving hydroxychloroquine were included in the primary analysis. The incidence of active SARS-CoV-2 infections during the study period did not differ between patients receiving hydroxychloroquine and patients not receiving hydroxychloroquine (31 [0·3%] of 10 703 vs 78 [0·4%] of 21 406; odds ratio 0·79, 95% CI 0·52-1·20, p=0·27). There were no significant differences in secondary outcomes between the two groups in patients who developed active SARS-CoV-2 infection. For all patients in the study, overall mortality was lower in the hydroxychloroquine group than in the group of patients who did not receive hydroxychloroquine (odds ratio 0·70, 95% CI 0·55-0·89, p=0·0031). In multivariate logistic regression analysis, receipt of hydroxychloroquine was not associated with the development of active SARS-CoV-2 infection (odds ratio 0·79, 95% CI 0·51-1·42). INTERPRETATION: Hydroxychloroquine was not associated with a preventive effect against SARS-CoV-2 infection in a large group of patients with rheumatological conditions. FUNDING: None.

Environmental trade-offs of pig production systems under varied operational efficiencies.

Production of pork, the most consumed meat globally, is estimated to emit 668 m tonnes CO2-eq of greenhouse gases each year. Amongst various production systems that comprise the pig industry, grain-based intensive production is widely regarded as the largest polluter of the environment, and thus it is imperative to develop alternative systems that can provide the right balance between sustainability and food security. Using an original dataset from the Republic of Ireland, this paper examines the life-cycle environmental impacts of representative pig farms operating under varying production efficiencies. For the baseline farm with an average production efficiency, global warming potential (GWP), acidification potential (AP) and eutrophication potential (EP) per kg carcass weight departing the slaughterhouse were estimated to be 3.5 kg CO2-eq, 43.8 g SO2-eq and 32.1 g PO4-eq, respectively. For herds with a higher production efficiency, a 9% improvement in feed conversion ratio was met by 6%, 15% and 12% decreases in GWP, EP, AP, respectively. Scenario and sensitivity analyses also revealed that (a) a switch to high-protein diets results in lower GWP and higher AP and EP, and (b) reducing transportation distances by sourcing domestically produced wheat and barley does not lower environmental impacts in any notable manner. To improve cross-study comparability of these findings, results based on an auxiliary functional unit, kg liveweight departing the farm gate, are also reported.

microRNAs with AAGUGC seed motif constitute an integral part of an oncogenic signaling network.

microRNA (miRNA) dysregulation is a common feature of cancer cells, but the complex roles of miRNAs in cancer are not fully elucidated. Here, we used functional genomics to identify oncogenic miRNAs in non-small cell lung cancer and evaluate their impact on response to epidermal growth factor (EGFR)-targeting therapy. Our data demonstrate that miRNAs with an AAGUGC motif in their seed sequence increase both cancer cell proliferation and sensitivity to EGFR inhibitors. Global transcriptomics, proteomics and target prediction resulted in the identification of several tumor suppressors involved in the G1/S transition as AAGUGC-miRNA targets. The clinical implications of our findings were evaluated by analysis of AAGUGC-miRNA expression in multiple cancer types, supporting the link between this miRNA seed family, their tumor suppressor targets and cancer cell proliferation. In conclusion, we propose the AAGUGC seed motif as an oncomotif and that oncomotif-miRNAs promote cancer cell proliferation. These findings have potential therapeutic implications, especially in selecting patients for EGFR-targeting therapy.

Amyloid-beta Alzheimer targets - protein processing, lipid rafts, and amyloid-beta pores.

Amyloid beta (Aß), the hallmark of Alzheimer's Disease (AD), now appears to be deleterious in its low number aggregate form as opposed to the macroscopic Aß fibers historically seen postmortem. While Alzheimer targets, such as the tau protein, amyloid precursor protein (APP) processing, and immune system activation continue to be investigated, the recent discovery that amyloid beta aggregates at lipid rafts and likely forms neurotoxic pores has led to a new paradigm regarding why past therapeutics may have failed and how to design the next round of compounds for clinical trials. An atomic resolution understanding of Aß aggregates, which appear to exist in multiple conformations, is most desirable for future therapeutic development. The investigative difficulties, structures of these small Aß aggregates, and current therapeutics are summarized in this review.

Attitudes towards mental health and help-seeking in railway workers.

BACKGROUND: TRiM (Trauma Risk Management) has been shown to improve mental health and attitudes towards mental health in high-risk occupational groups; however, there has been no research into how TRiM might work for railway workers. AIMS: To assess whether attending a TRiM training course alters mental health and attitudes to mental health-related help-seeking in railway workers. METHODS: Workers completed a survey assessing mental health and attitudes towards mental health and help-seeking, before and after a 2-day TRiM course; follow-up questionnaires were administered 4 months post-course. RESULTS: Fifty railway employees completed the questionnaires. Post-course scores for cohesion and mental health peer literacy (i.e. feeling able to recognize and discuss mental health symptoms with colleagues) and some aspects of stigma significantly improved, while there were non-significant improvements in common mental disorder and post-traumatic stress symptoms. The response rate for completing follow-up surveys was small (n = 8) but results from these subjects suggested mental health peer literacy scores remained significantly improved. CONCLUSIONS: This study provides a useful insight into attitudes of railway workers regarding stigma and their confidence in discussing trauma-related mental health. Significant improvements in cohesion and mental health peer literacy along with the general improvement in scores post-TRiM course provide some evidence of the potential benefits of TRiM training in railway workers. Follow-up results have limited reliability due to the small number of responders but suggest possible long-term benefits of attending a TRiM course. Further research is required to confirm this finding.

Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis.

Little is known about the alterations in microRNA (miRNA) expression patterns during the consecutive stages of cervical cancer development and their association with chromosomal instability. In this study, miRNA expression in normal cervical squamous epithelium, high-grade precancerous lesions (cervical intraepithelial neoplasia (CIN2-3)), squamous cell carcinomas (SCCs) and adenocarcinomas (AdCAs) was integrated with previously generated chromosomal profiles of the same samples. Significantly differential expression during the consecutive stages of cervical SCC development was observed for 106 miRNAs. Of these differentially expressed miRNAs, 27 showed early transiently altered expression in CIN2-3 lesions only, 46 miRNAs showed late altered expression in SCCs only and 33 showed continuously altered expression in both CIN2-3 and SCCs. Altered expression of five significantly differentially expressed miRNAs, hsa-miR-9 (1q23.2), hsa-miR-15b (3q25.32), hsa-miR-28-5p (3q27.3), hsa-miR-100 and hsa-miR-125b (both 11q24.1), was directly linked to frequent chromosomal alterations. Functional analyses were performed for hsa-miR-9, representing a potential oncogene with increased expression linked to a chromosomal gain of 1q. Hsa-miR-9 overexpression was found to increase cell viability, anchorage-independent growth and migration in vitro. Upon organic raft culturing, hsa-miR-9 hampered differentiation and induced proliferation in all strata of the epithelial layer. These findings support a potential oncogenic function of hsa-miR-9 in cervical cancer. In summary, differential expression of 106 miRNAs, partly associated with chromosomal alterations, was observed during cervical SCC development. Altered expression of hsa-miR-9 associated with a chromosomal gain of chromosome 1q was shown to be functionally relevant, underlining the importance of deregulated miRNA expression in cervical carcinogenesis.

An isoform of GTPase regulator DOCK4 localizes to the stereocilia in the inner ear and binds to harmonin (USH1C).

The driving forces for the regulation of cell morphology are the Rho family GTPases that coordinate the assembly of the actin cytoskeleton. This dynamic feature is a result of tight coupling between the cytoskeleton and signal transduction and is facilitated by actin-binding proteins (ABPs). Mutations in the actin bundling and PDZ domain-containing protein harmonin are the causes of Usher syndrome type 1C (USH1C), a syndrome of congenital deafness and progressive blindness, as well as certain forms of non-syndromic deafness. Here, we have used the yeast two-hybrid assay to isolate molecular partners of harmonin and identified DOCK4, an unconventional guanine exchange factor for the Rho family of guanosine triphosphatases (Rho GEF GTPases), as a protein interacting with harmonin. Detailed molecular analysis revealed that a novel DOCK4 isoform (DOCK4-Ex49) is expressed in the brain, eye and inner ear tissues. We have further provided evidence that the DOCK4-Ex49 binds to nucleotide free Rac as effectively as DOCK2 and DOCK4 and it is a potent Rac activator. By immunostaining using a peptide antibody specific to DOCK4-Ex49, we showed its localization in the inner ear within the hair bundles along the stereocilia (SC). Together, our data indicate a possible Rac-DOCK4-ABP harmonin-activated signaling pathway in regulating actin cytoskeleton organization in stereocilia.

Immunolocalization of P2Y4 and P2Y2 purinergic receptors in strial marginal cells and vestibular dark cells.

K+ secretion by strial marginal cell and vestibular dark cell epithelia is regulated by UTP and ATP at both the apical and basolateral membranes, suggesting control by P2Y2 and/or P2Y4 purinergic receptors. Immunolocalization was used to determine the identity and distribution of these putative receptors. Membrane proteins from gerbil brain, gerbil vestibular labyrinth and gerbil stria vascularis were isolated and analyzed by Western blot. P2Y2 antibody stained one band at 42 kDa for each tissue, whereas P2Y4 antibody stained 3 bands on gerbil brain (75, 55 and 36 kDa), one band on gerbil stria vascularis (55 kDa) and two bands on vestibular labyrinth (42 and 56 kDa). All bands were absent when the antibodies were blocked with their respective antigenic peptide. P2Y4 was immunolocalized by fluorescence confocal microscopy to only the apical membrane of strial marginal cells and vestibular dark cells and was similar to apical immunostaining of KCNE1 in the same cells. By contrast, P2Y2 was observed on the basolateral but not the apical membrane of dark cells. Similarly, in the stria vascularis P2Y2 was observed in the basolateral region but not the apical membrane of marginal cells. Additional staining was observed in the spiral ligament underlying the stria vascularis. These findings identify the molecular bases of the regulation of K+ secretion by apical and basolateral UTP in the inner ear.

Mutation in the beta-tubulin signature motif suppresses microtubule GTPase activity and dynamics, and slows mitosis.

We introduced a threonine-to-glycine point mutation at position 143 in the "tubulin signature motif" 140Gly-Gly-Gly-Thr-Gly-Ser-Gly146 of Saccharomyces cerevisiae beta-tubulin. In an electron diffraction model of the tubulin dimer, this sequence comes close to the phosphates of a guanine nucleotide bound in the beta-tubulin exchangeable E site. Both the GTP-binding affinity and the microtubule (MT)-dependent GTPase activity of tubulin isolated from haploid tub2-T143G mutant cells were reduced by at least 15-fold, compared to tubulin isolated from control wild-type cells. The growing and shortening dynamics of MTs assembled from alphabeta:Thr143Gly-mutated dimers were also strongly suppressed, compared to control MTs. The in vitro properties of the mutated MTs (slower growing and more stable) are consistent with the effects of the tub2-T143G mutation in haploid cells. The average length of MT spindles in large-budded mutant cells was only 3.7 +/- 0.2 microm, approximately half of the size of MT arrays in large-budded wild-type cells (average length = 7.1 +/- 0.4 microm), suggesting that there is a delay in mitosis in the mutant cells. There was also a higher proportion of large-budded cells with unsegregated nuclei in mutant cultures (30% versus 12% for wild-type cells), again suggesting such a delay. The results show that beta:Thr143 of the tubulin signature motif plays an important role in GTP binding and hydrolysis by the beta-tubulin E site and support the idea that tubulins belong to a family of proteins within the GTPase superfamily that are structurally distinct from the classic GTPases, such as EF-Tu and p21(ras). The data also suggest that MT dynamics are critical for MT function in yeast cells and that spindle MT assembly and disassembly could be coordinated with other cell-cycle events by regulating beta-tubulin GTPase activity.

Immunolocalization of ClC-K chloride channel in strial marginal cells and vestibular dark cells.

Secretion of K(+) into endolymph depends on a particular constellation of ion transport proteins in the apical and basolateral membranes of strial marginal cells and vestibular dark cells. One fundamental component is the large chloride conductance of the basolateral membrane, which recycles chloride taken up by the Na(+)-K(+)-Cl(-) cotransporter in the same membrane. Evidence has been reported recently that supports ClC-K, a channel subunit previously thought to be specific to the kidney, as being the molecular entity underlying this conductance. We have isolated protein from the gerbil kidney, stria vascularis and vestibular labyrinth and found by Western blot analysis a 60 kDa band, a 48 kDa band and 54 and 70 kDa bands, respectively, specifically labeled by ClC-K antibody. Subsequent immunohistochemical observations of the inner ear tissues with a confocal microscope on fluorescently labeled tissue sections showed the staining to be restricted to the basolateral region of strial marginal cells and vestibular dark cells. The cochlear staining was distinct from the distribution of the Kir4.1 (KCNJ10) K(+) channel, known to be present only in strial intermediate cells. These findings support the contention that ClC-K is an important component of the basolateral Cl(-) conductance that participates in K(+) secretion by these epithelia.

Distribution of frequenin in the mouse inner ear during development, comparison with other calcium-binding proteins and synaptophysin.

Frequenin is a calcium-binding protein previously implicated in the regulation of neurotransmission. We report its immunocytochemical detection in the mouse inner ear, in the adult, and during embryonic (E) and postnatal (P) development. The distribution of frequenin was compared with those of other calcium-binding proteins (calbindin, calretinin, parvalbumin) and synaptophysin. In the adult mouse inner ear, frequenin immunostaining was observed in the afferent neuronal systems (vestibular and cochlear neurons, their processes and endings) and in the vestibular and cochlear efferent nerve terminals. Frequenin colocalized with synaptophysin in well characterized presynaptic compartments, such as the vestibular and cochlear efferent endings, and in putative presynaptic compartments, such as the apical part of the vestibular calyces. Frequenin was not found in vestibular hair cells and in cochlear inner and outer hair cells. During development, frequenin immunoreactivity was first detected on E11 in the neurons of the statoacoustic ganglion. On E14, frequenin was detected in the afferent neurites innervating the vestibular sensory epithelium, along with synaptophysin. On E16, frequenin was detected in the afferent neurites below the inner hair cells in the organ of Corti. The timing of frequenin detection in vestibular and cochlear afferent neurites was consistent with their sequences of maturation, and was earlier than synaptogenesis. Thus in the inner ear, frequenin is a very early marker of differentiated and growing neurons and is present in presynaptic and postsynaptic compartments.

Molecular hashkeys: a novel method for molecular characterization and its application for predicting important pharmaceutical properties of molecules.

We define a novel numerical molecular representation, called the molecular hashkey, that captures sufficient information about a molecule to predict pharmaceutically interesting properties directly from three-dimensional molecular structure. The molecular hashkey represents molecular surface properties as a linear array of pairwise surface-based comparisons of the target molecule against a common 'basis-set' of molecules. Hashkey-measured molecular similarity correlates well with direct methods of measuring molecular surface similarity. Using a simple machine-learning technique with the molecular hashkeys, we show that it is possible to accurately predict the octanol-water partition coefficient, log P. Using more sophisticated learning techniques, we show that an accurate model of intestinal absorption for a set of drugs can be constructed using the same hashkeys used in the aforementioned experiments. Once a set of molecular hashkeys is calculated, its use in the training and testing of property-based models is very fast. Further, the required amount of data for model construction is very small. Neural network-based hashkey models trained on data sets as small as 30 molecules yield statistically significant prediction of molecular properties. The lack of a requirement for large data sets lends itself well to the prediction of pharmaceutically relevant molecular parameters for which data generation is expensive and slow. Molecular hashkeys coupled with machine-learning techniques can yield models that predict key pharmacological aspects of biologically important molecules and should therefore be important in the design of effective therapeutics.

Inactivity of N229A thymidylate synthase due to water-mediated effects: isolating a late stage in methyl transfer.

Mutation of thymidylate synthase N229(177) to alanine results in an essentially inactive enzyme, yet it leads to formation of a stable ternary complex. The kinetics of N229(177)A show that kcat for Escherichia coli is reduced by 200-fold while the Km for dUMP is increased 200-fold and the Km for folate increased by tenfold versus the wild-type enzyme. The crystal structures of N229(177)A in complex with dUMP and CB3717, and in complex with dUMP alone are determined at 2.4 A, and 2.5 A resolution. These structures identify the covalently bound ternary complex and show how N229(177)A traps an intermediate, and so becomes inactive in a later step of the reaction. Since the smaller alanine side-chain at N229(177)A does not directly sterically impair binding of ligands, the structures implicate, and place quantitative limits on the involvement of the structured water network in the active site of thymidylate synthase in both catalysis and in determining the binding affinity for dUMP (in contrast, the N229(177)V mutation in Lactobacillus casei has minimal effect on activity).

D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine.

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except cysteine abolish activity. We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual. CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes. The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form. In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites. In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor. In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor. This orientation blocks the conformational change required for forming covalent ternary complexes. Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring. Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis.

The additivity of substrate fragments in enzyme-ligand binding.

BACKGROUND: Enzymes have evolved to recognise their target substrates with exquisite selectivity and specificity. Whether fragments of the substrate--perhaps never available to the evolving enzyme--are bound in the same manner as the parent substrate addresses the fundamental basis of specificity. An understanding of the relative contributions of individual portions of ligand molecules to the enzyme-binding interaction may offer considerable insight into the principles of substrate recognition. RESULTS: We report 12 crystal structures of Escherichia coli thymidylate synthase in complexes with available fragments of the substrate (dUMP), both with and without the presence of a cofactor analogue. The structures display considerable fidelity of binding mode and interactions. These complexes reveal several interesting features: the cofactor analogue enhances the localisation of substrate and substrate fragments near the reactive thiol; the ribose moiety reduces local disorder through additional specific enzyme-ligand interactions; the pyrimidine has multiple roles, ranging from stereospecificity to mechanistic competence; and the glycosidic linkage has an important role in the formation of a covalent attachment between substrate and enzyme. CONCLUSIONS: The requirements of ligand-protein binding can be understood in terms of the binding of separate fragments of the ligand. Fragments which are subsystems of the natural substrate for the enzyme confer specific contributions to the binding affinity, orientation or electrostatics of the enzymatic mechanism. This ligand-binding analysis provides a complementary method to the more prevalent approaches utilising site-directed mutagenesis. In addition, these observations suggest a modular approach for rational drug design utilising chemical fragments.

An essential role for water in an enzyme reaction mechanism: the crystal structure of the thymidylate synthase mutant E58Q.

A water-mediated hydrogen bond network coordinated by glutamate 60(58) appears to play an important role in the thymidylate synthase (TS) reaction mechanism. We have addressed the role of glutamate 60(58) in the TS reaction by cocrystalizing the Escherichia coli TS mutant E60(58)Q with dUMP and the cofactor analog CB3717 and have determined the X-ray crystal structure to 2.5 A resolution with a final R factor of 15.2% (Rfree = 24.0%). Using difference Fourier analysis, we analyzed directly the changes that occur between wild-type and mutant structures. The structure of the mutant enzyme suggests that E60(58) is not required to properly position the ligands in the active site and that the coordinated hydrogen bond network has been disrupted in the mutant, providing an atomic resolution explanation for the impairment of the TS reaction by the E60(58)Q mutant and confirming the proposal that E60(58) coordinates this conserved hydrogen bond network. The structure also provides insight into the role of specific waters in the active site which have been suggested to be important in the TS reaction. Finally, the structure shows a unique conformation for the cofactor analog, CB3717, which has implications for structure-based drug design and sheds light on the controversy surrounding the previously observed enzymatic nonidentity between the chemically identical monomers of the TS dimer.